Transport of the HIV-1 pre-integration complex into the nucleus of infected cell is a critical step in virus replication. Therefore, compounds that target this step have great potential value as anti-HIV therapeutics. The parent grant of this application, R01 AI 40386, investigates two novel classes of anti-HIV compounds, arylene bis(methylketone) and naphtoquinone, with activity to inhibit HIV-1 nuclear import. To study the mechanism of action and effectiveness of these compounds, the parent grant relies on indirect biochemical analysis. Such approach has several limitations, including inability to study the activity of the drugs in live cells. In this application, we propose to apply modern imaging technology to monitor nuclear import of the HIV-1 pre-integration complex. Recent advances in fluorescent microscopic imaging, including new fluorophores and sensitive registration devices, make possible the viewing of individual virus-specific complexes in living cells for the first time. Two protocols for labeling pre-integration complexes will be used: incorporation of the fluorescent reporter molecule, green fluorescent protein (GFP), fused to the HIV-1 accessory protein, Vpr, into virion particles; and incorporation of fluorescent labeled nucleotides into the viral cDNA during reverse transcription. The latter technique will be used in two different modifications: nucleotides will be added to the virus either during endogenous reverse transcription (to produce highly labeled virus for co-localization studies) or during native reverse transcription (for live cell studies). The fate of fluorescent-labeled pre-integration complexes in infected macrophages will be followed by confocal microscopy. Live cell imaging techniques will provide an ultimate test for the activity and mechanism of action of the compounds investigated in the parent grant. The proposed research falls within the scope of this PAS as it uses new and innovative imaging modality to analyze cellular trafficking of the HIV-1 pre-integration complex. It is also consistent with the exploratory/development nature of the R21 mechanism, as it facilitates the application of this new technology to currently funded project. The imaging technology will greatly augment the value of the project by adding a superior method for analyzing the activity of the drugs.